bioRxiv preprint doi: https://doi.org/10.1101/315739; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Identification of TEX101 functional interactome through proteomic measurement of human spermatozoa homozygous for the missense variant rs35033974 Christina Schiza1,2, Dimitrios Korbakis1,3, Keith Jarvi3,4, Eleftherios P. Diamandis1,2,3,5* and Andrei P. Drabovich1,2,5* 1Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 2Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Canada 3Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada 4Department of Surgery, Division of Urology, Mount Sinai Hospital, University of Toronto, Toronto, Canada 5Department of Clinical Biochemistry, University Health Network, Toronto, Canada Correspondence should be addressed to: A. P. Drabovich, Ph.D., Department of Laboratory Medicine and Pathobiology, University of Toronto, 60 Murray Street; Room L6-201-4, Toronto, ON, M5T 3L9, Canada. Tel: (416) 586-4800 ext. 8805; e-mail: [email protected] or E. P. Diamandis, Ph.D., M.D., Mount Sinai Hospital, 60 Murray St [Box 32]; Flr 6 - Rm L6- 201-1, Toronto, ON, M5T 3L9, Canada. Tel: 416-586-8443; Fax: 416-619-5521; e-mail: [email protected] Non-standard abbreviations: TEX101, Testis-expressed sequence 101 protein; LY6K, Lymphocyte antigen 6 complex, locus K; ADAM29, A disintegrin and metalloproteinase domain- containing protein 29; DPEP3, Dipeptidase 3; BH-adjusted t-test, Benjamini-Hochberg-adjusted t-test; FDR, False discovery rate; FWHM, Full width at half maximum; GPI, Glycosylphosphatidylinositol; LC-MS/MS, liquid chromatography - tandem mass spectrometry; LFQ, Label-free quantification; MS, Mass spectrometry; mAb, Monoclonal antibody; MWU, Mann Whitney Unpaired t-test; PRM, Parallel reaction monitoring; ROC AUC, Receiver operating characteristic area under the curve; SCX, strong cation exchange chromatography; SP, seminal plasma; SNV, Single nucleotide variation; SRM, Selected reaction monitoring; WT, wild-type. Key words: TEX101; testis-expressed sequence 101 protein; LY6K; Lymphocyte antigen 6 complex, locus K; ADAM29, A disintegrin and metalloproteinase domain-containing protein 29; interactome; testis-specific proteins; spermatozoa; single nucleotide variant, unexplained male infertility; proteogenomics. 1 bioRxiv preprint doi: https://doi.org/10.1101/315739; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. SUMMARY TEX101 is a testis-specific cell-surface protein expressed exclusively in the male germ cells and a validated biomarker of male infertility. Mouse TEX101 was found essential for male fertility, and was suggested to function as a cell surface chaperone involved in maturation of proteins required for sperm migration and sperm-oocyte interaction. However, the precise functional role of human TEX101 is not known and cannot be studied in vitro due to the lack of human germ cell lines. Here, we genotyped 386 healthy fertile men and sub-fertile patients for a common and potentially deleterious missense variant rs35033974 of TEX101, and identified 52 heterozygous and 4 homozygous patients. We then discovered by targeted proteomics that the variant allele rs35033974 was associated with near-complete degradation (>97%) of the corresponding G99V TEX101 form, and suggested that spermatozoa of homozygous patients could serve as a knockdown model to study TEX101 function in humans. Differential proteomic profiling with label-free quantification measured 8,046 proteins in spermatozoa of eight men and identified 8 cell-surface and 9 secreted testis-specific proteins significantly down-regulated in four patients homozygous for rs35033974. Substantially reduced levels of testis-specific cell-surface proteins potentially involved in sperm migration and sperm-oocyte fusion (including LY6K and ADAM29) were confirmed by targeted proteomics and western blotting assays. Since recent population- scale genomic data revealed homozygous fathers with biological children, rs35033974 is not a single pathogenic factor of male infertility in humans. However, median TEX101 levels in seminal plasma were found 5-fold lower (P=0.0005) in heterozygous than in wild-type men of European ancestry. We conclude that spermatozoa of rs35033974 homozygous men have substantially reduced levels of TEX101 and could be used as a model to elucidate the precise TEX101 function, which will advance biology of human reproduction. 2 bioRxiv preprint doi: https://doi.org/10.1101/315739; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. INTRODUCTION Recent -omics studies identified 1,079 human genes with exclusive expression in testis (1). While function of many of testis-specific proteins is not known, it may be assumed that these proteins have unique and specialized roles in spermatogenesis and fertilization. Mutations, natural knockouts or deleterious single nucleotide variations in testis-specific genes could lead to severe male infertility with spermatogenesis arrest, low sperm concentration, abnormal spermatozoa morphology or low motility. In addition, mutations in testis-specific genes involved in sperm transit, acrosome reaction and sperm-oocyte interaction may result in no apparent phenotypical issues and male infertility which cannot be explained with routine diagnostics (2-4). We previously discovered and validated a testis-specific protein TEX101 as a seminal plasma biomarker for the differential diagnosis of azoospermia and male infertility (5-9). The functional role of TEX101 is not known, but based on mouse models it was suggested as a spermatozoa cell-surface chaperone involved in the maturation of four cell-surface proteins from the ADAM family (10-12). Tex101 knockout in mice resulted in absolute male sterility but normal sperm concentration, morphology and other phenotypical characteristics (10, 11). In the absence of TEX101 protein, ADAM 3-6 proteins were not properly processed and degraded. However, mouse data could not be translated into human studies since ADAM3, ADAM5 and ADAM6 genes are non- coding pseudogenes, while ADAM4 is not present in the human genome (13). Due to the lack of stable human male germ cell lines, TEX101-dependent proteins could not be identified in humans. As an alternative, we suggested that the functional role of TEX101 could be studied in human clinical samples, such as spermatozoa. Our previous work on TEX101 levels in seminal plasma revealed a small population of men with high sperm count, but unexplained infertility and very low levels of TEX101 protein in seminal plasma and spermatozoa (8). In this work, we hypothesized that some genomic alterations, such as natural knockouts or deleterious single nucleotide variations, could result in undetectable or low levels of TEX101 protein. We suggested that spermatozoa obtained from such patients could be used as knockout or knockdown models to identify proteins degraded in the absence of TEX101 and discover the functional interactome of TEX101 in humans. Unlike immunoprecipitation approaches to reveal direct and strong physical interactions, profiling of the whole proteome of spermatozoa lacking TEX101 could identify the whole functional interactome including weak and transients interactions. Collectively, such data could support in humans the previously suggested function of TEX101 as a cell-surface chaperone (10). 3 bioRxiv preprint doi: https://doi.org/10.1101/315739; this version posted May 7, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. EXPERIMENTAL PROCEDURES Study design and statistical rationale The objectives of this study were to identify potential genomic alterations which could impact levels of TEX101 protein, and verify those levels experimentally in human spermatozoa samples. We suggested that differential proteomic profiling of spermatozoa with such genomic alterations could identify proteins degraded in the absence of TEX101 and thus discover the functional interactome of TEX101 in humans. According to power calculations, differential profiling of spermatozoa of 4 wild-type and 4 rs35033974 homozygous men could identify proteins down-regulated at least 2.4-fold, assuming 80% power, α = 0.05, 1.8% coefficient of variation for log2-transfromed LFQ intensity values and a one-tailed t-test. Power calculations were done with G*Power software (version 3.1.7, Heinrich Heine University Dusseldorf). GraphPad Prism (v5.03) was used to generate scatter plots, perform statistical analysis, and calculate the area under the Receiver Operating Characteristic curve (ROC AUC). Non-parametric Mann – Whitney U test was used to compare TEX101 levels in seminal